The invention relates generally to vitronectin-derived cell culture substrates and methods of using the same for culturing pluripotent stem cells.
Pluripotent stem cells, such as embryonic stem (ES) cells and induced pluripotent stem (iPS) cells, are characterized by self-renewal without differentiation and the ability to differentiate into cells of all three germ layers (Evans & Kaufman, Nature 292:154-156 (1981)). An important aspect of culturing pluripotent stem cells are the culture conditions themselves. Successful maintenance and experimental use of pluripotent stem cells includes culture conditions that provide growth factors and appropriate substrates to sustain viability and pluripotency.
Pluripotent stem cell culture methods have evolved considerably in an effort to define conditions and reduce inter-culture variability. Completely-defined growth media, such as TESR™ (Ludwig et al., Nat. Methods 3:637-646 (2006)), were developed to provide controllable and reproducible sources of basic nutrients and growth factors for survival and expansion of pluripotent cells to directly determine how pluripotent stem cells grow and differentiate. However, experimental variability is introduced by the use of varying and undefined substrates. According to conventional culture methods, pluripotent stem cells are grown on a layer of feeder cells or on complex artificial matrices, such as MATRIGEL™. Both feeder layers and complex matrices fluctuate unpredictably in their composition. Because the precise composition of these matrices cannot be determined, it is difficult to predict how the substrate interacts with the cell or media components.
To reduce this compositional variability, pluripotent cells have recently been cultured on isolated or recombinant extracellular matrix proteins (Miyazaki et al., Biochem. Biophys. Res. Commun. 375:27-32 (2008); Braam et al., Stem Cells 26:2257-2265 (2008)). Examples of extracellular matrix proteins successfully used as substrates for pluripotent cell culture include laminin, fibronectin, E-cadherin, and vitronectin.
Vitronectin has been successfully employed as an in vitro substrate for many cell types, including human pluripotent stem cells. Immature vitronectin is converted to its mature form when a 19-amino acid signal peptide at its N-terminus is cut off during the process of protein maturation to form mature vitronectin. Mature vitronectin, herein referred to as vitronectin, is a 459 amino acid glycoprotein of approximately 75 kDa that contains an amino-terminal domain (N-terminal amino acids 1-44), which includes a somatomedin B domain, followed by a Arg-Gly-Asp (RGD) sequence, a central domain rich in hydrophobic amino acids (central amino acids 131-342), and a carboxyl-terminal domain (C-terminal amino acids 380-459) The N-terminal somatomedin B domain (SMB) and the C-terminal V10 domain are functional domains in vitronectin.
The high cost and low yield associated with producing vitronectin in animal cell cultures limit use of the protein in animal cell culture methods. Also, vitronectin production from animal cell culture bears the inherent risk of animal protein contamination. While vitronectin can be produced by recombinant methods, recombinant vitronectin has shown very low activity in cell culture.
Pluripotent stem cells are most useful for research and clinical application when the conditions used to derive and culture them are fully defined and controlled. Accordingly, there is a need in the art for substrates free of components that introduce inconsistencies to maintain control over pluripotent cell culture conditions. Specifically, there is a need in the art for pluripotent cell culture substrates containing only those components that support pluripotent cell function. Further, the art seeks fully-defined substrates that can be produced easily, cheaply, and in large quantities without contamination of animal protein.